PPAR compounds for use in the treatment of fibrotic diseases

ABSTRACT

The invention relates to the use of a pan-PPAR agonist, or of a pharmaceutical composition containing said agonist, for the treatment of a fibrotic condition.

FIELD OF THE INVENTION

The present invention relates to the use of a pan-PPAR agonist or of apharmaceutical compositors containing said agonist, for the treatment offibrotic diseases.

BACKGROUND OF THE INVENTION

Liver fibrosis is the result of a complex interplay among different celltypes. It is characterized by the recruitment of inflammatory cells inresponse to chronic injury and by the activation of hepatic stellatecells (HSCs), leading to the accumulation of extracellular matrix.Steatosis is commonly coexisting with hepatic inflammation andhepatocellular injury. Increased oxidative stress is a common factor inall chronic liver diseases leading to fibrosis, regardless of theiretiology. Injured hepatocytes, HSCs, and infiltrating inflammatory cellsare major sources of reactive oxygen species (ROS). Indeed, theoxidative stress will induce the recruitment of inflammatory cells andthe activation of HSCs. Therefore, in a chronic liver injury context, avicious circle of hepatocyte damage, ROS production, HSC activation, andinflammatory cell recruitment will occur, amplifying the fibrogenicanswer to injury.

Means for an effective treatment for liver fibrotic diseases, such asnon-alcoholic fatty liver disease (NAFLD) and non-alcoholicsteatohepatitis (NASH), are still insufficient. No treatment isestablished for patient with NASH, and several therapeutic options aretested in clinical trial (Vuppalanchi R and Chalasani N, Hepatology2009, 49(1): 306-317; Dowman J. K et al., Q. J. Med. 2010,103(2):71-83). These studies involve the use of many different familiesof chemical compounds (fibrates, thiazolidinediones, biguanides,statins, cannabinoids) and therapeutic targets (nuclear receptors,angiotensin receptors, cannabinoid receptors, HMG-CoA reductase).Recently, studies involving thiazolidinediones (rosiglitazone andpioglitazone) have shown that these drugs may improve liver conditionbut treatment with these drugs is not without undesired effects such ashigher risks of congestive cardiac failure and osteoporosis, as well asweight gain with psychological effects on the patient (Dowman J. K etal., op. cit.; Shiri-Sverdlov R et al., J. Hepatol. 2006 44: 732-41;Neuschwander-Tetri et al., Hepatology 2003, 38:1008-1017). Clinicaltrials involving the administration of cannabinoids have raised theconcern of neuropsychiatric disruption (Vuppalanchi R and Chalasam N,op. cit.). Other therapies currently ongoing are seeking to assess inNASH drugs as antioxidants but none of these treatments has yet showedconvincing results (Nelson A et al., J. Clin. Gastroenterol 2009, 43:990-994). Candidates for the treatment of liver diseases are disclosedin WO 2011/064350 and US 2013/108573. There is still a need, however,for compounds which are suitable for the treatment of liver diseases, inparticular for compounds which may target the several components offibrotic process such as steatosis, inflammation and collagen depositionand are devoid of the side effects observed with the drugs currentlyunder evaluation.

Chronic kidney disease (CKD), also known as chronic renal disease, is aprogressive loss in renal function over a period of months or years. CKDhas its general meaning in the art and is used to classify numerousconditions that affect the kidney, destruction of the renal parenchymaand the loss of functional nephrons or glomeruli. It should be furthernoted that CKD can result from different causes, but the final pathwayremains renal fibrosis. Examples of etiology of CKD include, but are notlimited to, cardiovascular diseases, hypertension, diabetes,glomerulonephritis, polycystic kidney diseases, and kidney graftrejection. Renal fibrosis, characterized by glomerulosclerosis andtubuloininterstitial fibrosis, is the common manifestation of a widevariety of chronic kidney diseases. The pathogenesis of renal fibrosisis, in essence, a monotonous process that is characterized by anexcessive accumulation and deposition of extracellular matrix (ECM)components. Renal fibrosis is a progressive process that ultimatelyleads to end-stage renal failure, a devastating disorder that requiresdialysis or kidney transplantation. However, there is no specifictreatment unequivocally shown to slow the worsening of chronic kidneydisease. Injury to the kidney is associated with release ofcytokines/growth factors such as TGF-β, epidermal growth factor (EGF),and platelet derived growth factor (PDGF) by damaged or infiltratingcells. An increase in production of TGF-β is one of the most importantmechanisms in the pathogenesis of renal fibrogenesis. TGF-β1 stimulatesfibroblast cell activation and induces matrix expression through itsinteraction with TGF-β receptors, which are mainly composed of twoprotein families—type I (TβRI) and type II (TβRII) receptors. TGF-β1binds to TβRII, which results in TβRI recruitment to form a heteromericTGF-β receptor complex. The complex phosphorylates and activates Smad2and Smad3, the two major Smads that mediate the profibrotic events.Other signaling pathways such as extracellular regulated kinase 1/2(ERK1/2) can also be activated in response to TGF-β receptor activation.Activated ERK1/2 contributes to tubular cell apopiosis in theobstructive kidney. Since actuation of TCP-β signaling is considered tobe the major mechanism that directly promotes fibroblast activation andfibrosis progression, therapeutic intervention of this pathway could beconsidered as a strategy to halt or prevent renal fibrosis. Candidatesfor the treatment of CKD are disclosed in WO 2012/159107 and WO2014/013005. There is still a need, however, for compounds which aresuitable for the treatment of CKD.

Lung fibrotic remodelling occurs in pulmonary disease conditions such asacute respiratory distress syndrome, chronic obstructive pulmonarydisease and asthma. Pulmonary fibrosis is characterised by the excessivedeposition of extracellular matrix in the interstitium, resulting inrespiratory failure. Pulmonary fibrosis can be caused by a number ofdifferent conditions, including sarcoidosis, hypersensitivitypneumonitis, collagen vascular disease, and inhalant exposure. In asignificant number of patients, no underlying cause for the pulmonaryfibrosis can be found. These conditions of unknown etiology have beentermed idiopathic interstitial pneumonias. The most common form ofidiopathic interstitial pneumonia is idiopathic pulmonary fibrosis(IPF). The primary histopathologic finding of IPF is that of usualinterstitial pneumonia with temporal heterogeneity of alternating zonesof interstitial fibrosis with fibroblastic foci (i.e., newer fibrosis),inflammation, honeycomb changes (i.e., older fibrosis), and normal lungarchitecture (i. e., no evidence of fibrosis). Candidates for thetreatment of IPF are disclosed in WO 2004/103296. Candidates for thetreatment of pulmonary fibrotic disorders are disclosed in WO2009/149188. Recently, studies involving thiazolidinediones such asrosiglitazone have shown that these drugs may improve pulmonary fibrosisbut treatment with these drugs is not without undesired effects such ashigher risks of congestive cardiac failure (Kung J et al., Expert Opin.Drug Saf. 2012, 11(4):565-579). Pirfenidone(5-methyl-1-phenyl-2-(1H)-pyridone) has anti-fibrotic properties and isapproved in Europe and Japan for the treatment of IPF. There is still aneed, however, for alternative compounds which are suitable for thetreatment of pulmonary fibrotic disorders.

Fibrotic disorders are characterized by abnormal and excessivedeposition of collagen and other extracellular matrix (ECM) componentsin various tissues. Although their aetiology is quite diverse, thepresence of ECM-producing fibroblasts displaying an activated phenotypein the affected tissues is typical of fibrotic diseases. Fibroblastactivation is characterized by a marked increase in the transcriptionalactivity of the genes encoding type I and type III collagens andfibronectin, initiation of the expression of alpha-smooth muscle actin(α-SMA), and the reduction of ECM degradative activities. The mostfrequent systemic fibrotic disorder is systemic fibrosis which is a ratechronic disease of unknown cause. It is a clinically heterogeneous,systemic disorder which affects the connective tissue of the skininternal organs and the walls of blood vessels. It is characterized byalterations of the microvasculature, disturbances of the immune systemand by massive deposition of collagen and other matrix substances in theconnective tissue. Basic functions of various cell types (endothelialcells, T-lymphocytes, monocytes, fibroblasts, mast cells) as well as theproduction and effects of cytokines, growth factors, and adhesionmolecules are known to be involved in the development of this disease.Systemic fibrosis is often referred to as scleroderma. The spectrum ofsclerodermatous diseases comprises a wide variety of clinical entitiessuch as morphea (patchy, linear, and generalized), pseudo-sclerodermaand the overlap-syndromes with similar cutaneous and histopathologicmanifestations. In addition, the complex pathophysiology of systemicfibrosis, involving genetic factors, environmental factors, vascular andimmune system functions, as well as fibroblasts and matrix substances,and the complexity of the internal organ involvement, results insclerodermatous diseases often being studied as autoimmune or connectivetissue diseases. Therefore, systemic fibrosis has been a challenge forclinicians with regards to diagnostic procedures and therapeuticregimens. Clinical diagnosis of systemic fibrosis often involvesattention from several disciplines (e.g. dermatologists,rheumatologists, pulmonologists, nephrologists, and gastroenterologists)and may include invasive procedure such as a biopsy of the fibrotictissue and/or skin for confirmation. Candidates for the treatment ofsystemic fibrosis are disclosed in US 2013/0287794 and US 2014/0038956.There is still a need however, for alternative compounds which aresuitable for the treatment of systemic fibrosis.

The peroxisome proliferator-activated receptors (PPARs) are a group ofnuclear receptor proteins that function as transcription factorsregulating the expression of genes. PPARs play essential roles in theregulation of cellular differentiation, development, and metabolism(carbohydrate, lipid, protein). Three subtypes of PPARs have beenidentified:

-   -   PPARα, which are mainly expressed in liver, kidney, heart,        muscle, adipose tissue and lungs;    -   PPARγ, which are expressed in virtually all tissues;    -   PPARδ, which are observed on a variety of tissues/cells notably        in the cardiovascular, urinary, respiratory, digestive and        musculoskeletal systems.

PPAR agonists are drugs which act upon the PPARs. They are used for thetreatment of symptoms of the metabolic diseases, mainly for loweringtriglycerides and blood sugar. PPARα agonists essentially consist of theclass of fibrates (e.g. fenofibrate). PPARγ agonists essentially consistof thiazolidinediones (e.g. rosiglitazone and pioglitazone). PPARδagonists include GW501516, a candidate compound that was eventuallydiscontinued due to safety issues.

PPAR receptors expression is modified in fibrosis diseases. For example,decrease expression of PPARγ has been reproducibly described in skinbiopsies, as well as in explanted skin fibroblasts from systemicscleroderma patients (Lakota et al, Arthritis Res. Ther. 2012 May 1;14(3)). A lower expression of PPARγ was also reported in lungfibroblasts from scleroderma patients (Bogatkevich et al, Pulm. Med. Vol2012; 2012). PPARγ agonists rosiglitazone and pioglitazone protectrodents from bleomycin-induced skin and lung fibrosis in vivo andprevent activation of profibrolic pathways and processes in vitro infibroblast cell lines and in primary fibroblasts (Aoki et al,Respiration. 2009:77(3):311-9; Samah et al, Eur J Pharmacol. 2012 Aug.15; 689(1-3)). PPARα receptors also modulate the profibrolic response todifferent stimuli. In the lung, fenofibrate, a specific PPARα agonist,prevented bleomycin-induced fibrosis (Samah et al 2012 op cit).Furthermore, PPARδ agonist GW0742 has been shown to reduce lunginflammation induced by bleomycin instillation in mice (Galuppo et al.Int J Immunopathol Pharmacol. 2010 October-December; 23(4):1033-46).

SUMMARY OF THE INVENTION

It has now been found that pan-PPAR agonists, i.e. compounds whichactivate all three PPAR receptors (PPARα, PPARγ and PPARδ), exertbeneficial effects in the treatment of various fibrotic conditions. Thepresent invention therefore provides a pan-PPAR agonist for use in amethod of treatment of a fibrotic condition. The invention also providescompositions and methods for treating a fibrotic condition.

In one embodiment, the fibrotic condition is a condition affecting anyorgan which can develop fibrosis, such as the heart, the lung, theliver, the kidney, the gastrointestinal tract, the skin, etc.

In another embodiment, the fibrotic condition is selected from: liverfibrosis, fatty liver disease, non-alcoholic steatohepatitis, chronickidney disease, a pulmonary fibrotic disorder such as idiopathicpulmonary fibrosis, and systemic scleroderma.

In yet another embodiment, which can be combined with the previous ones,the pan-PPAR agonist is intended tor oral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasma, triglycerides levels of CCl₄-exposed mice treatedwith vehicle, compound A and rosiglitazone.

FIG. 2 shows collagen deposition in CCl₄-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 3 shows TGFβ-1 expression in CCl₄-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 4 shows Col1a expression in CCl₄-exposed mice treated with vehicle,compound A and rosiglitazone.

FIG. 5 shows α-SMA expression in CCl₄-exposed mice treated with vehicle,compound A and rosiglitazone.

FIG. 6 shows MCP-1 expression in CCl₄-exposed mice treated with vehicle,compound A and rosiglitazone.

FIG. 7 shows Fibronectin expression in CCl₄-exposed mice treated withvehicle, compound A and rosiglitazone.

Legend to FIGS. 1 to 7: in the oil group the bars represent, from leftto right, vehicle, compound A (100 mg/kg) and rosiglitazone, in the CCl₄group the bars represent, from left to right, vehicle compound A (30mg/kg) compound A (100 mg/kg) and rosiglitazone.

FIG. 8 shows serum urea levels of anti-GBM exposed mice treated withvehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 9 shows urinary volumes of anti-GBM exposed mice treated withvehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 10 shows urinary albumin levels of anti-GBM exposed mice treatedwith vehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 11 shows osteopontin levels of anti-GBM exposed mice treated withvehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 12 shows MCP-1 levels of anti-GBM exposed mice treated withvehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 13 shows TGFβR1 expression, of anti-GBM exposed mice treated withvehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 14 shows Col1a expression of anti-GBM exposed mice treated withvehicle, captopril, rosiglitazone, pioglitazone and compound A.

FIG. 15 shows Col3a expression of anti-GBM exposed mice treated withvehicle, captopril rosiglitazone, pioglitazone and compound A.

FIG. 16 shows the number of pathologies glomeruli in anti-GBM exposedmice treated with vehicle and compound A.

Legend to FIGS. 8 to 15: the bars represent, from left to right, controlmice, anti-GBM exposed mice treated with vehicle, anti-GBM exposed micetreated with captopril anti-GBM exposed mice treated with rosiglitazone,anti-GBM exposed mice treated with pioglitazone, anti-GBM exposed micetreated with compound A (30 mg/kg) and anti-GBM exposed mice treatedwith compound A (100 mg/kg). Legend to FIG. 16: the bars represent, fromleft to right, anti-GBM exposed mice treated with vehicle and anti-GBMexposed mice treated with compound A (100 mg/kg).

FIG. 17 shows collagen deposition in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 18 shows TIMP-1 levels in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 19 shows MCP-1 levels in bleomycin-exposed. mice treated withvehicle, compound A and rosiglitazone.

FIG. 20 shows osteopontin levels in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 21 shows TGFβR1 expression in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 22 shows Col1a expression in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 23 shows Col3a expression in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 24 shows TIMP-1 expression in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 25 shows MCP-1 expression in bleomycin-exposed mice treated withvehicle, compound A and rosiglitazone.

FIG. 26 shows osteopontin expression in bleomycin-exposed mice treatedwith vehicle, compound A and rosiglitazone.

FIG. 27 shows Fibronectin expression in bleomycin-exposed mice treatedwith vehicle, compound A and rosiglitazone.

Legend to FIGS. 17 to 27: in the saline group the bars represent, fromleft to right, vehicle, compound A (100 mg/kg) and rosiglitazone; in thebleomycin group the bars represent, from left to right, vehicle,compound A (30 mg/kg), compound A (100 mg/kg) and rosiglitazone.

FIG. 28 shows the dermal thickness of bleomycin exposed mice treatedwith vehicle, compound A and rosiglitazone.

FIG. 29 shows the hydroxyproline content in bleomycin-exposed micetreated with vehicle, compound A and rosiglitazone.

FIG. 30 shows the collagen content in bleomycin-exposed mice treatedwith vehicle, compound A and rosiglitazone.

Legend to FIGS. 28 to 30: Bleo=bleomycin; IVA30=compound A (30 mg/kg);IVA100=compound A (100 mg/kg); Ros=rosiglitazone.

FIG. 31 shows the activation of the PPARα, γ and δ human receptors bycompound A as a function of the concentration of said compound.

FIG. 32 shows the activation of the PPARα, γ and δ murine receptors bycompound A as a function of the concentration of said compound.

FIG. 33 shows the effect of compound A, fenofibric acid androsiglitazone on PDGF-induced proliferation in primary human lungfibroblasts.

FIG. 34 shows the effect of compound A, fenofibric acid androsiglitazone on PDGF-induced proliferation in primacy human dermalfibroblasts.

FIG. 35 shows the effect of compound A, fenofibric acid androsiglitazone on TGFβ-induced FMT in primary human lung fibroblasts.

FIG. 36 shows the effect of compound A, fenofibric acid androsiglitazone on TGFβ-induced FMT in primary human dermal fibroblasts.

Legend to FIGS. 33 to 36: Rosi=rosiglitazone; Feno=fenofibric acid.

In FIGS. 1 to 36, compound A is5-Chloro-1-[(6-benzothiazolyl)sulfonyl]-1H-indole-2-butanoic acid.

DETAILED DESCRIPTION OF THE INVENTION

Chronic liver injury caused by fats, alcohol, virus or chemicalsubstance may induce the activation of hepatic stellate cell forsecreting a large amount of extracellular matrix such as collagen, whichmay lead to liver fibrosis as a consequence of the extracellular matrixover-deposition.

Chronic kidney disease (CKD) is the result of various insults so thekidney, affecting approximately 10% of the normal population. It is aprogressive process marked by interstitial fibrosis. The primary aim oftreatment in patients with CKD is to prevent or at least to slowprogression of CKD.

Pulmonary fibrosis also called idiopathic pulmonary fibrosis (IPF),interstitial diffuse pulmonary fibrosis, inflammatory pulmonaryfibrosis, or fibrosing alveolitis, is an inflammatory lung disorder anda heterogeneous group of conditions characterized by abnormal formationof fibrous tissue between alveoli caused by alveolitis comprising aninflammatory cellular infiltration into the alveolar septae withresulting fibrosis. The effects of IPF are chronic, progressive, andoften fatal. A number of investigations about pulmonary fibrosis haveindicated that sustained and augmented expression of some cytokines inthe lung are relevant to recruitment of inflammatory cells andaccumulation of extracellular matrix components followed by remodelingof the lung architecture. In particular, proinflammatory cytokines suchas TNF-α and interleukin IL-1β were demonstrated to play major roles inthe formation of pneumonitis and pulmonary fibrosis. In addition,profibrotic cytokines such as TGF-α and CTGF also play critical roles inthe pathogenesis of pulmonary fibrosis.

Scleroderma is a disease that causes thickened skin and varying degreesof organ dysfunction resulting from small-vessel vasculopathy andimmune-mediated fibrosis. The clinical manifestations of this diseaseare extremely heterogeneous and depend on the presence and degree ofinternal organ involvement. Patients can present with a spectrum ofillness ranging from localized skin fibrosis only (localizedscleroderma) to a systemic disorder with both cutaneous and internalorgan involvement. Localized scleroderma includes various forms ofcutaneous sclerosis without internal organ involvement. These forms ofscleroderma can be disfiguring but only rarely require systemic therapyto control disease activity. Systemic sclerosis is further divided intotwo subsets of disease, depending on the degree of skin and organinvolvement. The presence of diffuse systemic sclerosis denotes thepresence of extensive cutaneous sclerosis over the proximal limbs,trunk, and face. Patients with limited systemic sclerosis have fibrosislimited to the hands, forearms, feet, legs and face. Both diffuse andlimited systemic sclerosis are associated with internal organinvolvement; however, patients with diffuse systemic sclerosis are atgreater risk of clinically significant major organ dysfunction. Somepatients with limited systemic sclerosis may be further classified ashaving the CREST syndrome, with accompanying calcinosis, Raynaud'sphenomenon, esophageal dysmotility, sclerodactyly, and cutaneoustelangiectasias. Scleroderma since sclerosis is a rare disorder in whichpatients develop vascular and fibrotic damage to internal organs in theabsence of cutaneous sclerosis. The pathophysiology of systemicsclerosis involves vascular damage and activation of fibroblasts, andcollagen and other extracellular proteins in various tissues areoverproduced. Scleroderma is characterized by immune system activation,endothelial dysfunction, and enhanced fibroblast activity. The preciseinciting events leading to the development of systemic sclerosis arecurrently unknown. Several cytokines including interleukin-4 andtransforming growth factor-beta (TGF-β) have been implicated infibroblast activation in patients with scleroderma. These cytokines arereleased from activated immune cells, fibroblasts, and endothelialcells. Activated fibroblasts elaborate structurally normal collagen andother extracellular matrix proteins in the skin and various internalorgans.

The present invention is based on the finding that a pan-PPAR agonistexerts beneficial effects in the treatment of the fibrotic conditionssuch as those described above. In the context of the present invention,the term “pan-PPAR agonist” is intended to mean a compound whichsignificantly activates each of the PPARα, PPARγ and PPARδ receptors,i.e. a compound which would individually be regarded as a PPARα agonist,a PPARγ agonist, and a PPARδ agonist based on its respective EC50values. According to the present invention, significant activation ofthe PPARα, PPARγ and PPARδ receptors is achieved when the EC50 for eachreceptor is ≤10⁻⁶ M. The EC50s for the three receptor subtypespreferably differ by less than 2 orders of magnitude (i.e. the ratio ofthe EC50 for two receptor subtypes is either less than 100 or greaterthan 0.01). In one embodiment the pan-PPAR agonist is not bezafibrate.

In one aspect, the invention therefore provides a pan-PPAR agonist foruse in the treatment of a fibrotic condition.

In one embodiment, the fibrotic condition is a condition affecting anyorgan which can develop fibrosis, such as the heart, the lung, theliver, the kidney, the gastrointestinal tract, the skin, etc.

In a further embodiment, the fibrotic condition is selected from: liverfibrosis, fatty liver disease, non-alcoholic steatohepatitis, chronickidney disease, a pulmonary fibrotic disorder, systemic scleroderma.

In yet a further embodiment, the fibrotic condition is a liver disease,preferably liver fibrosis, fatty liver disease, or non-alcoholicsteatohepatitis.

In yet a further embodiment, the fibrotic condition is chronic kidneydisease. The disease is notably selected from nephropathy (e.g.membranous nephropathy, diabetic nephropathy and hypertensivenephropathy), glomerulonephritis (e.g. membranous glomerulonephritis andmembranoproliferative glomerulonephritis such as rapidly progressiveglomerulonephritis), interstitial nephritis, lupus nephritis, idiopathicnephrotic syndrome (e.g. minimal change nephrotic syndrome and focalsegmental glomerulosclerosis), obstructive uropathy, polycystic kidneydisease (e.g. Autosomal Dominant Polycystic Kidney Disease and AutosomalRecessive Polycystic Kidney Disease), and kidney graft rejection (e.g.acute and chronic kidney rejection).

In yet a further embodiment, the fibrotic condition is a pulmonaryfibrotic disorder, preferably idiopathic pulmonary fibrosis.

In yet a further embodiment, the fibrotic condition is a skin fibrosissuch as systemic scleroderma.

In yet a further embodiment, which can be combined with any of theprevious embodiments, the pan-PPAR agonist is intended for oraladministration.

The pan-PPAR agonist can be formulated into a pharmaceutical compositionfor administration.

In another aspect, the invention therefore provides a pharmaceuticalcomposition including a pan-PPAR agonist, together with apharmaceutically acceptable excipient, for use in the treatment of afibrotic condition as described above in the various embodiments of thefirst aspect of the invention. In one embodiment, the compositioncomprises a therapeutically effective amount of a pan-PPAR agonist. Inthe context of the invention, the term “therapeutically effectiveamount” means a sufficient amount of pan-PPAR agonist to provide thedesired effect. Ultimately, the attending physician decides theappropriate amount and dosage regimen.

In yet another aspect the invention provides the use of a pan-PPARagonist in the manufacture of a medicament for the treatment of afibrotic condition as described above in the various embodiments of thefirst aspect of the invention.

In yet another aspect, the invention provides a method of treating afibrotic condition as described above in the various embodiments of thefirst aspect of the invention, which comprises administering to asubject in need thereof a therapeutically effective amount of a pan-PPARagonist. The subject is typically a mammal, preferably a human. The term“therapeutically effective amount” has the same meaning as mentionedabove.

The pan-PPAR agonist will generally be administered as a formulation inassociation with one or more pharmaceutically acceptable excipients. Theterm ‘excipient’ is used herein to describe any ingredient other thanthe pan-PPAR agonist. The choice of excipient(s) will to a large extentdepend on factors such as the particular mode of administration, theeffect of the excipient on solubility and stability, and the nature ofthe dosage form.

Pharmaceutical compositions suitable for the delivery of the pan-PPARagonist and methods for that preparation will be readily apparent tothose skilled in the art. Such compositions and methods for theirpreparation may be found, for example, in Remington's PharmaceuticalSciences, 19th Edition (Mack Publishing Company, 1995), incorporatedherein by reference.

Oral Administration

The pan-PPAR agonist may be administered orally. Oral administration mayinvolve swallowing, so that the compound enters the gastrointestinaltract, and/or buccal, lingual, or sublingual administration by which thecompound enters the blood stream directly from the mouth. Formulationssuitable for oral administration include solid, semi-solid and liquidsystems such as tablets; soft or hard capsules containing multi- ornano-particulates, liquids, or powders; lozenges (includingliquid-filled); chews; gels; fast dispersing dosage forms; films;ovules; sprays; and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsules(made, for example, from gelatin or hydroxypropylmethylcellulose) andtypically comprise a carrier, for example, water, ethanol, polyethyleneglycol, propylene glycol, methylcellulose, or a suitable oil, and one ormore emulsifying agents and or suspending agents. Liquid formulationsmay also be prepared by the reconstitution of a solid, for example, froma sachet.

For tablet or capsule dosage forms, depending on dose, the drug may makeup from 1 weight % to 80 weight % of the dosage form, more typicallyfrom 5 weight % to 60 weight % of the dosage form. In addition to thedrug, tablets generally contain a disintegrant. Examples ofdisintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, croscarmellose sodium,crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystallinecellulose, lower alkyl-substituted hydroxypropyl cellulose, starch,pregelatinised starch and sodium alginate. Generally, the disintegratewill comprise from 1 weight % to 25 weight %, preferably from 5 weight %to 20 weight % of the dosage form.

Binders are generally used to impart cohesive qualities to a tabletformulation. Suitable binders include microcrystalline cellulose,gelatin, sugars, polyethylene glycol, natural and synthetic gums,polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose andhydroxypropyl methylcellulose. Tablets may also contain diluents, suchas lactose (monohydrate, spray-dried monohydrate, anhydrous and thelike), mannitol xylitol, dextrose, sucrose, sorbitol, microcrystallinecellulose, starch and dibasic calcium phosphate dihydrate.

Tablets or capsules may also optionally comprise surface active agents,such as sodium lauryl sulfate and polysorbate 80, and glidants such assilicon dioxide and talc. When present, surface active agents maycomprise from 0.2 weight % to 5 weight % of the tablet, and glidants maycomprise from 0.2 weight % to 1 weight % of the tablet.

Tablets also generally contain lubricants such as magnesium stearate,calcium stearate, zinc stearate, sodium stearyl fumarate, and mixturesof magnesium stearate with sodium lauryl sulphate. Lubricants generallycomprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight %to 3 weight % a of the tablet.

Other possible ingredients include anti-oxidants, colorants, flavoringagents, preservatives and taste-masking agents.

Exemplary tablets contain up to about 80 weight % drug, from about 10weight % to about 90 weight % binder, from about 0 weight % to about 85weight % diluent, from about 2 weight % to about 10 weight %disintegrant, and from about 0.25 weight % to about 10 weight %lubricant.

Tablet blends may be compressed directly or by roller to form tablets.Tablet blends or portions of blends may alternatively be wet-, dry-, ormelt-granulated, melt congealed, or extruded before tableting. The finalformulation may comprise one or more layers and may be coated oruncoated; it may even be encapsulated.

Parenteral Administration

The pan-PPAR agonist may also be administered directly into the bloodstream, into muscle, or into an internal organ. Suitable means forparenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular, intrasynovial andsubcutaneous. Suitable devices for parenteral administration includeneedle (including microneedle) injectors, needle-free injectors andinfusion techniques.

Parenteral formulations are typically aqueous solutions which maycontain excipients such as salts, carbohydrates and buffering agents(preferably to a pH of from 3 to 9), but, for some applications, theymay be more suitably formulated as a sterile non-aqueous solution or asa dried form to be used in conjunction with a suitable vehicle such assterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, forexample, by lyophilisation, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The solubility of the pan-PPAR agonist used in the preparation ofparenteral solutions may be increased by the use of appropriateformulation techniques, such as the incorporation ofsolubility-enhancing agents or technologies like SMEDDS (Self MicroEmulsifying Drug Delivery System).

Formulations for parenteral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease. The pan-PPAR agonist may be formulated as a suspension or as asolid, semi-solid, or thixotropic liquid for administration as animplanted depot providing modified release of the active compound.Examples of such formulations include drug-coated stents and semi-solidsand suspensions comprising drug-loaded poly(dl-lactic-coglycolic)acid(PGLA) microspheres.

Inhaled/Intranasal Administration

The pan-PPAR agonist may also be administered intranasally or byinhalation, typically in the form of a dry powder (either alone, as amixture, for example, in a dry blend with lactose, or as a mixedcomponent particle, for example, mixed with phospholipids, such asphosphatidylcholine) from a dry powder inhaler, as an aerosol spray froma pressurised container, pump, spray, atomiser (preferably an atomiserusing electrohydrodynamics to produce a fine mist), or nebuliser, withor without the use of a suitable propellant, such as1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, or asnasal drops. For intranasal use, the powder may comprise a bioadhesiveagent, for example, chitosan or cyclodextrin.

The pressurized container, pump, spray, atomizer, or nebuliser containsa solution or suspension of pan-PPAR agonist comprising, for example,ethanol, aqueous ethanol, or a suitable alternative agent fordispersing, solubilising, or extending release of the active, apropellant(s) as solvent and an optional surfactant, such as sorbitantrioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug productis micronized to a size suitable for delivery by inhalation (typicallyless than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenisation, or spray drying.

Capsules (made, for example from gelatin orhydroxypropylmethylcellulose), blisters and cartridges for use in aninhaler or insufflator may be formulated to contain a powder mix ofpan-PPAR agonist, a suitable powder base such as lactose or starch and aperformance modifier such as l-leucine, mannitol, or magnesium stearate.The lactose may be anhydrous or in the form of the monohydrate,preferably the latter. Other suitable excipients include dextran,glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

A suitable solution formulation for use in an atomiser usingelectrohydrodynamics to produce a fine mist may contain from 1 μg to 20mg of pan-PPAR agonist per actuation and the actuation volume may varyfrom 1 μl to 100 μl. A typical formulation may comprise the pan-PPARagonist, propylene glycol, sterile water, ethanol and sodium chloride.Alternative solvents which may be used instead of propylene glycolinclude glycerol and polyethylene glycol.

Suitable flavours, such as menthol and levomenthol, or sweeteners, suchas saccharin or saccharin sodium, may be added to those formulations ofthe invention intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release using, for example, PGLA. Modifiedrelease formulations include delayed-, sustained-, pulsed-, controlled-,targeted and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit isdetermined by means of a valve which delivers a metered amount. Units inaccordance with the invention are typically arranged to administer ametered dose or “puff” containing from 1 μg to 10 mg of pan-PPARagonist. The overall daily dose will typically be in the range 1 μg to200 mg which may be administered in a single dose or, more usually, asdivided doses throughout the day.

Topical Administration

The pan-PPAR agonist may also be administered topically,(intra)dermally, or transdermally, to the skin or mucosa. Typicalformulations for this purpose include gels, hydrogels, lotions,solutions, creams, ointments, dusting powders, dressings, foams, films,skin patches, wafers, implants, sponges, fibres, bandages andmicroemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol and propylene glycol. Penetrationenhancers may be incorporated.

Other means of topical administration include delivery byelectroporation, iontophoresis, phonophoresis, sonophoresis andmicroneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.

Formulations for topical administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

Oral and parenteral administrations are suitable irrespective of thetype of fibrotic condition. Topical administration is suitable when thefibrotic condition is e.g. systemic scleroderma. Inhaled/intranasaladministration is suitable when the fibrotic condition is e.g. pulmonaryfibrosis or systemic scleroderma.

For oral administration, the pan-PPAR agonist can be administered to apatient at dosage levels in the range of from about 100 mg to about3,000 mg per day, preferably, from about 500 mg to about 3,000 mg perday. The total daily dose may be administered in single or divideddoses. A pharmaceutical composition according to the invention maytypically contain from about 100 to about 1000 mg of pan-PPAR agonist,for example 100, 200, 500, 750 or 1000 mg of pan-PPAR agonist.

Typically suspensions of pan-PPAR agonist in 1% methylcellulose solutionand in 1% methylcellulose+0.5% poloxamer were prepared. Capsulescontaining 25, 50 or 200 mg of pan-PPAR agonist were also prepared. IVformulations where the pan-PPAR agonist is dissolved in 30%/70% w/wPEG400/0.05 M phosphate buffer, pH 8 (25-100 μg/ml) were also prepared.

In yet a further embodiment, which can be combined with any of theprevious embodiments or any of the aspects of the invention, thepan-PPAR agonist is5-Chloro-1-[(6-benzothiazolyl)sulfonyl]-1H-indole-2-butanoic acid (alsoreferred to as “compound A”). Compound A and its method of preparationare described in WO 2007/026097. It has been found that compound Aactivates each of the PPARα, PPARγ and PPARδ receptors. Compound A canbe used in the context of the present invention in the form of one ofits pharmaceutically acceptable salts or solvates. The term ‘solvate’ isused herein to describe a molecular complex comprising compound A andone or more pharmaceutically acceptable solvent molecules, for example,ethanol. The term ‘hydrate’ is employed when said solvent is water.Pharmaceutically acceptable salts of compound A include the acidaddition and base salts thereof.

Suitable acid addition salts are formed from acids which form non-toxicsalts. Examples include the acetate, adipate, aspartate, benzoate,besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate,citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate,gluconate, glucuronate, hexafluorophosphate, hibenzate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,isethionate, lactate, malate, maleate, malonate, mesylate,methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate,oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate,tartrate, tosylate, trifluoroacetate and xinofoate salts.

Suitable base salts are formed from bases which form non-toxic salts.Examples include the aluminium, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine and zinc salts. Hemisalts of acids andbases may also be formed, for example, hemisulphate and hemicalciumsalts.

Pharmaceutically acceptable salts of compound A may be prepared by oneor more of three methods:

(i) by reacting the compound with the desired acid or base;

(ii) by removing an acid- or base-labile protecting group from asuitable precursor of the compound or by ring-opening a suitable cyclicprecursor, for example, a lactone or lactam, using the desired acid orbase; or

(iii) by converting one salt of the compound to another by reaction withan appropriate acid or base or by means of a suitable ion exchangecolumn.

All three reactions are typically carried out in solution. The resultingsalt may precipitate out and be collected by filtration or may berecovered by evaporation of the solvent. The degree of ionisation in theresulting salt may vary from completely ionised to almost non-ionised.

The invention is illustrated by the following examples.

Example 1: Effect of Compound A on the Development of CarbonTetrachloride-Induced Liver Fibrosis in Mice, and Comparison with KnownPPARγ Agonist

It has been reported (Yao T et al., Am J Physiol. 1994 September; 267(3Pt 1):G476-84) that carbon tetrachloride (CCl₄) induces hepatocytemitochondrial dysfunction and oxidative stress in a mouse model, leadingto collagen deposition and liver fibrosis. The effect of compound A androsiglitazone, a known PPARγ agonist, has accordingly been assessed in amurine model of CCl₄-induced liver fibrosis.

Mice were daily orally treated tor 22 days with compound A at twodifferent doses (30 and 100 mg/kg/day) and with the PPARγ referencecompound rosiglitazone at 5 mg/kg/day. At the end of the treatment,animals were sacrificed and plasma samples and livers were harvested.Collagen deposition and expression of genes that are known to beinvolved in liver inflammation and fibrosis were quantified and somerelated plasmatic biomarkers were measured.

CCl₄-exposed mice orally treated with vehicle for days displayed ahepatic fibrosis as shown by the statistically significant increase incollagen level in liver tissue. Treatment with compound A significantlyreduced hepatic fibrosis by 80% (30 mg/kg/day) and 89% (100 mg kg day),respectively and improved many of the related markers. Treatment withrosiglitazone reduced hepatic fibrosis by 54% only while most markerswere either unchanged or even worsened.

MATERIALS AND METHODS

The experiments were carried out using 56 male C57BL/6J mice (JANVIERLABS, C.S. 4105, Saint-Berthevin, France), weighing 21-24 g at thebeginning of the experiment. The animals were housed in groups of 3-10in polypropylene cages (floor area=1032 cm²) under standard conditions:room temperature (22±2° C.), hygrometry (55±10%), light/dark cycle (12h/12 h), air replacement (15-20 volumes/hour), water and food (SDS, RM1)ad libitum. Mice were allowed to habituate for at least 5 days prior toexperimentation. Mice were numbered by marking their tail usingindelible markers.

Ready-to-use suspensions of compound A (3 mg ml and 10 mg/mL) androsiglitazone (0.5 mg/mL) were stored at 5±3° C. Ready-to-useformulations of vehicle (methyl cellulose 400 cP 1%+0.1% Poloxamer 188)were also stored at 5±3° C. Carbone tetrachloride (CCl₄) (Sigma Chemicalco, Saint Quentin Fallavier, France) was freshly prepared each day ofdosing in sunflower oil (v/v, 1/11).

Dosing

Mice were allocated to the following groups:

-   -   1. Sunflower oil (twice a week for 3 weeks, ip)/vehicle (once a        day for 22 days po), n=7,    -   2. Sunflower oil (twice a week for 3 weeks, ip)/compound A (100        mg/kg/d once a day for 22 days po), n=8,    -   3. Sunflower oil (twice a week for 3 weeks, ip)/rosiglitazone (5        mg/kg/d once a day for 22 days po), n=8    -   4. CCl₄ (3.5 mL/kg, twice a week for 3 weeks, ip)/vehicle (once        a day for 22 days po), n=8    -   5. CCl₄ (3.5 mL/kg, twice a week for 3 weeks, ip)/compound A (30        mg/kg/d once a day for 22 days po), n=8,    -   6. CCl₄ (3.5 mL/kg, twice a week for 3 weeks, ip)/compound A        (100 mg/kg/d once a day for 22 days po), n=7    -   7. CCl₄ (3.5 mL/kg, twice a week for 3 weeks, ip)/rosiglitazone        (5 mg/kg/d once a day for 22 days po), n=8.

Two days a week for 3 weeks, mice were intraperitoneally administered inthe morning with either 100 μL of CCl₄ (3.5 mL kg in sunflower oil (v/v,1/11)) or 100 μL of sunflower oil. In parallel, mice were orally treatedonce a day for 22 days (day 0 to day 21) with vehicle, compound A orrosiglitazone. On days with concomitant administrations of vehicle,compound A or rosiglitazone and CCl₄, vehicle, compound A orrosiglitazone was administered 6 hours before sunflower oil or CCl₄administration. The volume of administration of test compounds was 10mL/kg body weight for oral administrations.

Terminal Blood Sampling

On day 21, 2 hours after dosing, animals were anaesthetised withpentobarbital (60 mg/kg, ip) and blood was collected using cardiacpuncture. The exact time of dosing and time of sample collection werenoted for each animal. Blood sampling (0.9 ml, of total blood) wasplaced in pre-chilled 2-mL lithium-heparin collection tubes. The bloodsamples were gently mixed, placed on crushed ice and centrifuged within30 min of sampling at approximately 1500×g for 10 min at approximately+4° C. For each blood sampling, the resultant plasma was separated into2 aliquots (at least 100 μL each) and transferred using disposableplastic material into polypropylene tubes. The samples were immediatelytransferred in the upright position to a freezer where they were kept at20° C.

After terminal blood sampling, liver tissue was removed:

-   -   A first tissue sample (about 50 mg) was harvested and fixed with        paraformaldehyde, and at 5±3° C.,    -   A second tissue sample (200 mg) was frozen in liquid nitrogen        and kept at −20° C.

Measured Parameters

Collagen

For the quantification of collagen, sections were stained withPicro-Sirius red and counterstained with Mayer's haemoatoxylin. Allslides were digitized and 5 non overlapping fields of 3 differentsections were randomly analysed by means of the image J software(version 1.42, N.I.H., USA). For all slides, analysis was performed by asingle experimenter strictly in the same conditions.

Gene Expression

mRNA extraction was performed on small frozen liver samples (50-100 mg).Briefly, samples were cryogenically ground with mortar and pestle.Samples were subsequently homogenized using marble (2×5 mn) and 1 ml ofQiazol lysis reagent (Qiagen Ref 79306) in a Retsch MM300 apparatus. RNAextraction on liver homogenates was finalized with Qiagen Rneasy lipidKit (Ref 74804) according to the manufacturer's instructions. RNAquantify was determined with Nanodrop (ND2000 Thermo Scientific) and RNAquality was verified with Bioanalyzer (2100 Agilent Technology).

Random-primed cDNA synthesis was carried out on 100 ng total RNA usingthe Iscript kit (BIORAD ref 170-8891) according to the manufacturer'sinstructions Real-time PCR was carried out with 7.5 ng RNA equivalentson an ABI Prism 7900 Sequence Detection System (APPLIED BIOSYSTEMS)using Iq ITaq SYBR Green Universal Rox (Ref 1725124 Biorad) and usingdedicated QPCR primers. For some mRNA transcripts, quantification wasperformed using TaqMan probes labeled with the fluorochrome FAM andusing Universal PCR MasterMix No AmpErase UNG (APPLIED BIOSYSTEMS ref4324020). The primers used for the assays are listed in the followingtable:

Target Name Full target name Primer names Sequences (5′-3′) Rplp0ribosomal protein, Fw2Rplp0 PE ctgatgggcaagaacaccat (SEQ ID NO: 1)large, P0 Rev2Rplp0 PE gtgaggatcctccttggtgaa (SEQ ID NO: 2) Tgfb1transforming growth MTgfb1FW accggcccttcctgctcctc (SEQ ID NO: 3)factor, beta 1 MTgfb1REV gccgcacacagcagttcttc (SEQ ID NO: 4) Col1a1collagen, type 1, MCol1a1FW aaaggtgctgatggttctcc (SEQ ID NO: 5) alpha 1MCol1a1REV gggaccgggaggaccactgg (SEQ ID NO: 6) Fn1 fibronectin 1 MFn1FWgttgtctgacgctggctttaag (SEQ ID NO: 7) MFn1REVcccacttctctccgatcttgta (SEQ ID NO: 8) Acta 2 Actin, alpha 2, Macta2FWcagggagtaatggttggaatg (SEQ ID NO: 9) (α-SMA) smooth muscle, aortaMacta2REV tttccatgtcgtcccagttg (SEQ ID NO: 10) Ccl2Chemokine (C C motif) MCcl2FW aggtccctgtcatgcttctg (SEQ ID NO: 11)(MCP-1) ligand 2 MCcl2REV gcctactcattgggatcatc (SEQ ID NO: 12)Real Time PCR was performed on ABI PRISM 7900 apparatus Raw data fromABI7900 were exported in text format. Analysis was performed on Excel,the relative quantity of transcript were calculated using the “DeltaDelta CT method” (Livak et al. Methods 2001), using Rp1p0 ashousekeeping gene tor normalization and mean data from the non-treatedanimals (vehicle group) as reference control. Each RNA samples werereverse transcribed and quantified in triplicate.

Liver Biomarkers

Protocol of Liver Proteins Extraction:

Extraction with T-PER®: Tissue Protein Extraction Reagent, prod#78510(lot: NG174004), Thermo Scientific with: Halt Protease InhibitorSingle-Use Cocktail, EDTA-free (100×)—Prod#78425—Lot #NL178051 ThermoFischer.

Samples of liver and supernatants were kept on ice during allexperiment. For extraction, the manufacturer recommends 10 mg of tissuefor 100 μl T-Per+1 μl “Halt-protease” (100×).

50 ml of T-Per buffer were prepared to which 500 μl of “Halt-protease”(100×) were added, and the mixture was kept on ice. Samples of 50 to 70mg of liver just thawed were weighed and cut into small pieces, and 1 mlof cold PBS was added to wash the tissue. The mixture was centrifuged at500 g during 5 min at 4° C., and the supernatant was discarded. 100μl/10 mg of T-Per+antiproteases (100×) were added, crushing the liverwith a Potter, with 5 or 6 twists, up and down. The mixture wascentrifuged at 10000 g during 5 min at 4° C. The supernatant wasremoved, aliquoted and kept at −20° C. for the subsequent measurement ofbiomarkers. Samples of 10 μl were used to dose proteins by the BCAtechnique, after dilution 1/10^(ěme) in H₂O mq following the proceduredescribed in MOS: BAP-05-062-01 (Kit BCA—Pierce BCA protein assaykit—Pierce Thermo scientific, Ref: 23225).

All proteins were quantified with ELISA Kit, according to theinstructions of the manufacturers:

TIMP-1: Mouse TIMP-1, R&D SYSTEMS®, Ref: TM100

TGF-β1: Quantikine Mouse/Rat/Porcine Canine TGF-β1 immunoassay, R&DSYSTEMS®, ref: MB100B

Data Processing and Statistical Analysis

All parameters were analysed using Graph pad software (version 5.1). Theparameters were analysed as followed:

-   -   Using student's t test for independent samples to compare group        1 versus group 4 to validate the experiment (effect of CCl₄).    -   Using student's t test for independent samples to compare group        t versus group 2 and group 1 versus group 3 to investigate the        effect of compound A or rosiglitazone alone,    -   Using one-way ANOVA (treatment) to compare group 4 versus        compound A treated groups (5, 6) to investigate the effect of        compound A on CCl₄-induced liver fibrosis. As ANOVA was found        significant, a Dunnett's test was used,    -   Using student's t test for independent samples to compare group        4 versus rosiglitazone treated-group (group 7) to investigate        the effect of rosiglitazone on CCl₄-induced liver fibrosis.

In FIGS. 1 to 7, * denotes a p-value<0.05; ** denotes a p-value<0.01;*** denotes a p-value<0.001.

RESULTS

1/Plasma Triglycerides

In CCl₄-exposed mice, compound A (30 and 100 mg/kg/day) significantlyreduced plasma triglycerides compared to vehicle (p<0.05 and p<0.01,respectively) whereas rosiglitazone increased plasma triglycerideswithout teaching significance (FIG. 1).

2/Collagen Deposition

In CCl₄-exposed mice compound A (30 and 100 mg/kg/day) significantlydecreased collagen levels compared to vehicle (˜80%, p<0.01 and p<0.001,respectively), and rosiglitazone significantly decreased collagen levels(−54%, p<0 (FIG. 2).

3/TGFβ-1 Expression

In CCl₄-exposed mice, compound A (30 and 100 mg/kg/day) significantlyinhibited the expression of TGFβ-1 compared to vehicle (p<0.01 andp<0.001, respectively) whereas rosiglitazone significantly upregulatedthe expression of TGFβ-1 (p<0.001) (FIG. 3).

4/Collagen Type I, Alpha I (Col1a) Expression

In CCl₄-exposed mice, compound (30 and 100 mg/kg/day) significantlyinhibited the expression of Col1a compared to vehicle (p<0.05 andp<0.001, respectively) whereas rosiglitazone upregulated the expressionof Col1a without teaching significance (FIG. 4).

5/α-SMA Expression

In CCl₄-exposed mice, compound A (100 mg/kg/day) inhibited theexpression of α-SMA compared to vehicle without reaching significancewhereas rosiglitazone upregulated the expression of α-SMA withoutreaching significance (FIG. 5).

6/MCP-1 Expression

In CCl₄-exposed mice, compound A (100 mg/kg/day) significantly inhibitedthe expression of MCP-1 compared to vehicle (p<0.05) whereasrosiglitazone had no effect (FIG. 6).

7/Fibronectin Expression

In CCl₄-exposed mice, compound A (30 and 100 mg/kg/day) significantlyinhibited the expression of fibronectin compared to vehicle (p<0.001)whereas rosiglitazone upregulated the expression of fibronectin withoutreaching significance (FIG. 7).

The above results show that oral administration of compound A (30 or 100mg/kg/day) for 22 days in the male C57BL/6J mouse reduced CCl₄-inducedliver fibrosis. A dose-dependent effect was obtained with compound and amaximal 90% decrease in collagen levels was observed with compound A at100 mg/kg/day and improved significantly most of the associated markers.In contrast, rosiglitazone had a much less marked effect while at mosthaving no effect on other markers.

Example 2: Effect of Compound A on the Development of Anti-GBM InducedGlomerulonephritis in Mice, and Comparison with Known PPARγ Agonists andwith a Known ICE

Anti-GBM (Glomerular Basement Membrane) induced glomerulonephritis inmice is a commonly used in vivo model to evaluate the potential of newchemical entities against CKD). The effect of compound A ofrosiglitazone and pioglitazone (known PPARγ agonists), and of captopril(a known ICE—inhibitor of the (angiotensin) conversion enzyme), hasaccordingly been assessed in such a model.

Mice were daily orally treated for 7 days with compound A at twodifferent doses (and 100 mg/kg/day), with the PPARγ reference compoundsrosiglitazone (at 3 mg/kg/day) and pioglitazone (at mg/kg/day), and withthe reference ICE compound captopril at 10 mg/kg/day. At the end of thetreatment, animals were sacrificed and plasma samples and kidneys wereharvested. Expression of genes that are known to be implicated in CKDwas quantified and plasmatic levels of kidney parameters were measured.

Anti-GBM exposed mice orally treated with vehicle for 7 days displayed aglomerulonephritis and a fibrosis as shown by the statisticallysignificant increase of urinary urea, albuminuria, kidney osteopontin,kidney MCP-1 protein levels Col1 and Col3 expression compared tovehicle. Treatment with compound A in 30 and 100 mg/kg/day for 7 daysreduced urinary urea, albuminuria, kidney osteopontin, kidney MCP-1levels, Col1 and Col3 mRNA expression computed to vehicle (withsignificance at 100 mg/kg/day). In comparison, captopril had an effectsimilar to that of compound A on urinary urea, albuminuria, kidneyosteopontin, kidney MCP-1 protein levels but did not significantlyreduce Col1 and Col3 expression; rosiglitazone and pioglitazone,however, significantly increased kidney osteopontin and kidney MCP-1levels compared to vehicle.

MATERIALS AND METHODS

The experiments were carried out using female C5BL/6J mice (CERJJanvier, route des Chênes secs, Le Genest Saint Isle—France), aged 8-10weeks at the beginning of the experiment. The animals were randomlyassigned to polypropylene boxes (floor area=1032 cm²) by groups of 3-4,based on weight. Mice were individually marked on the ear. They wereallowed to habituate for a week prior to experimentation under standardconditions: room temperature (22±2° C.), hygrometry (55±10%), light/darkcycle (12 h/12 h), air replacement (15-20 volumes/hour), water and food(SDS, RM1) ad libitum.

Ready-to-use suspensions of compound A (3 mg/mL and 10 mg/mL,corresponding to a dose of 30 mg/kg and 100 mg/kg, respectively) invehicle [1% Methylcellulose (METOLOSE SM400, 400 Cps)+0.1% Poloxamer188] were stored at 5° C. until use.

Ready-to-use suspensions of rosiglitazone (0.3 mg/mL, corresponding to adose of 3 mg/kg) in vehicle were stored at 5° C. until use.

Ready-to-use suspensions of pioglitazone (3 mg/mL, corresponding to adose of 30 mg/kg) in vehicle were stored at 5° C. until use.

Ready-to-use suspensions of captopril (1 mg/mL, corresponding to a doseof 10 mg/kg) in vehicle were stored at 5° C. until use.

Sheep IgG antibody (ab37385, Abcam) preparation was carried out at 4° C.on ice. The antibody was dissolved in physiological serum so as toobtain a 4 mg/ml solution. Separately, complete Freund's adjuvant (CFA)was homogenized with a vortex. A 5 ml luer lock syringe was filled with2.5 ml of CFA. Another 5 ml luer lock syringe was filled with 2.5 ml ofSheep IgG solution. Both syringes were connected with a micro emulsionneedle taking care to remove all air bubbles. Sheep IgG solution waspassed in CFA. The mixture between the two syringes was repeatedlyforced during 5 minutes until a noticeable increase of resistance wasobserved. The micro emulsion needle was then replaced by an inox couplerluer female/luer female. The mixture between the two syringes was againforced during a few minutes. The surface tension in water was thentested. 1 ml luer lock syringes were filled, taking care to remove airbubbles. A 23G needle was added that the syringes were stored at 4° C.until use.

Sheep Anti-Rat Glomeruli (GBM) Serum ((PTX-001S, Probetex) was stored at5° C. until use.

Dosing

Mice were allocated to the following groups:

Immunization Challenge Emulsion anti- Mice Sheep GBM Group numberIgG/HR37Ra antibody Treatment 1 10 200 μg/500 μg no Vehicle 2 10 300 μlVehicle 3 10 Captopril 10 mg/kg 4 10 Rosiglitazone 3 mg/kg 5 10Pioglitazone 30 mg/kg 6 10 Compound A 30 mg/kg 7 10 Compound A 100 mg/kg5 days before anti-GBM antibody administration, animals were immunizedby subcutaneous injection on three sites (one over each hip and one inthe scruff of the neck) of 100 μl of Sheep IgG/CFA emulsion (200 μg ofSheep IgG/100 μg of Mycobacterium tuberculosis H37Ra), underO₂/isoflurane (1 L/3%) anesthesia.

On day 0, mice were weighed and placed under a heat lamp ramp for about20-30 minutes to allow vasodilatation of caudal veins and bettervisibility tor injection. Each animal was restrained in an injectioncone. Mice were given an intravenous injection of 300 μl of anti-GBMantibody serum. Mice were then orally administered with compound A,rosiglitazone, pioglitazone and captopril, and treatment was continuedonce a day (in the morning) for a further 7 days. Mice were weighedapproximately every other day.

On day 6 mice were weighed and transferred into a diuresis box wherethey stayed for 24 h. Bibs were weighed before and after diuresis inorder to measure the amount of water taken. On day 7 mice were weighed,received oral treatment and were returned in their initial box. Urineswere collected, centrifuged, measured and several aliquots were frozenat −80′T for subsequent assays (urea, creatinine, albumin . . . ).

In the afternoon of day 7 mice were anesthetized with a mixtureO₂/isoflurane 1 L/3%) and blood sampling was performed, in theretroorbitary sinus, with a Pasteur pipette. 400 μl of whole blood weretransferred in a dry microtube. Sera were obtained after 30 min ofclotting and 2 centrifugations at 6000 rpm for 15 minutes, at 4° c. Serawere aliquoted and frozen at −80° C. for subsequent assays (urea,creatinine, proteins, adiponectin). Then, mice were euthanized bycervical dislocation, and kidneys were harvested and weighed. The cortexof one kidney per mouse was isolated. Small pieces were kept forsubsequent RNA expression analysis as detailed below. The rest wasdirectly frozen in liquid nitrogen for subsequent assays (TGFbeta-1,OPN, MCP1 . . . ). Of the remaining kidney, one half was placed in anindividual histological cassette in 10% buffered formalin for 24 hoursfor histological analysis.

Measured Parameters

Gene Expression

mRNA extraction was performed on small frozen kidney samples (50-100mg). Briefly, samples were cryogenically ground with mortar and pestle.Samples were subsequently homogenized using marble (2×5 mn) and 1 ml ofQiazol lysis reagent (Qiagen Ref 79306) in a Retsch MM300 apparatus. RNAextraction on kidney homogenates was finalized with Qiagen Rneasy lipidKit (Ref 74804) according to the manufacturer's instructions. RNAquantity was determined with Nanodrop (ND2000 Thermo Scientific) and RNAquality was verified with Bioanalyzer (2100 Agilent Technology).

Random-primed cDNA synthesis was carried out on 100 ng total RNA usingthe Iscript kit (BIORAD ref 170-8891) according to the manufacturer'sinstructions. Real-time PCR was carried out with 7.5 ng RNA equivalentson an ABI Prism 7000 Sequence Detection System (APPLIED BIOSYSTEMS)using Iq ITaq SYBR Green Universal Rox (Ref 1725124 Biorad) and usingdedicated QPCR primers. For some mRNA transcripts, quantification wasperformed using TaqMan probes labeled with the fluorochrome FAM andusing Universal PCR MasterMix No AmpErase UNG (APPLIED BIOSYSTEMS ref4324020). The primers used for the assays are listed in the followingtable:

Target Name Full target name Primer names Sequences (5′-3′) Rplp0ribosomal protein, Fw2Rplp0 PE ctgatgggcaagaacaccat (SEQ ID NO: 1)large, P0 Rev2Rplp0 PE gtgaggtcctccttggtgaa (SEQ ID NO: 2) Tgfbr1transforming growth MTgfbr1FW ggtcttgcccatcttcacat (SEQ ID NO: 13)factor, beta receptor 1 MTgfbr1REVcaacaggttccatttttcttca (SEQ ID NO: 14) Col1a1 collagen, type 1,MCol1a1FW aaaggtgctgatggttctcc (SEQ ID NO: 5) alpha 1 MCol1a1REVgggaccgggaggaccactgg (SEQ ID NO: 6) Col3a1 collagen, type III, MCol3a1FWgggatcaaatgaaggcgaat (SEQ ID NO: 15) alpha 1 MCol3a1REVtgggtagtctcattgccttgc (SEQ ID NO: 16)Real Time PCR was performed on ABI PRISM 7900 apparatus Raw data fromABI7900 were exported in text format. Analysis was performed on Excel,the relative quantity of transcript were calculated using the “DeltaDelta CT method” (Livak et al. Methods 2001), using Rp1p0 ashousekeeping gene for normalization and mean data from the non-treatedanimals (vehicle group) as reference control. Each RNA samples werereverse transcribed and quantified in triplicate.

Urine, Plasma and Kidney Biomarkers

Serum and urinary assays (urea, creatinine, albumin, and proteins) werecarried out with a Konelab apparatus and corresponding colorimetrictests.

Micro-albuminuria was assayed with a fluorescent kit: Albumin BlueFluorescent Assay (Active Motif, Ref: 15002).

All kidney proteins were quantified with ELISA Kit, according to theinstructions of the manufacturers:

-   -   Osteopontin: Quantikine Mouse Osteopontin immunoassay, R&D        SYSTEMS®, Ref: MOST00    -   MCP-1: Quantikine Mouse CCL2/JE/MCP-1 ELISA Kit, R&D SYSTEMS®,        ref: MJE00

Data Processing and Statistical Analysis

All parameters were analysed using Graphpad software (version 5.1). Theparameters were analysed using one-way ANOVA (treatment) to comparegroups (1, 2, 3, 4, 5, 6 and 7). When ANOVA was found significant, aDunnett's test was used to compare group 2 to all other groups.

In FIGS. 8 to 16, * denotes a p-value<0.05; ** denotes a p-value<0.01;*** denotes a p-value<0.001.

RESULTS

1/Serum Urea

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyreduced urea levels compared, to vehicle (p<0.01) whereas captoprilreduced urea level without reaching significance and both rosiglitazoneand pioglitazone increased urea levels without reaching significance(FIG. 8).

2/Urinary Volume

In anti-GBM-exposed mice, compound A (30 and 100 mg/kg/day) reducedurinary volume compared to vehicle without reaching significance,whereas captopril also reduced urinary volume without reachingsignificance, and rosiglitazone and pioglitazone each increased urinaryalbumin without reaching significance (FIG. 9).

3/Urinary Albumin

In anti-GBM-exposed mice, compound A (100 mg/kg/day) reduced albuminlevels compared to vehicle without reaching significance, whereascaptopril also reduced albumin levels without reaching significance,rosiglitazone increased albumin levels without reaching significance,and pioglitazone significantly increased albumin levels (p<0.001) (FIG.10).

4/Kidney Osteapontin

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyreduced osteopontin levels compared to vehicle (p<0.05), whereascaptopril also significantly reduced osteopontin levels (p<0.05), androsiglitazone and pioglitazone each significantly increased osteopontinlevels (p<0.01 and p<0.05, respectively) (FIG. 11).

5/Kidney MCP-1

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyreduced MCP-1 levels compared to vehicle (p<0.001), whereas captoprilalso significantly reduced MCP-1 levels (p<0.01), and rosiglitazone andpioglitazone each significantly increased MCP-1 levels (p<0.05) (FIG.12).

6/TGFβR-1 Expression

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyinhibited TGFβR-1 expression compared to vehicle (p<0.01), whereascaptopril had no effect on TGFβ-R1 expression, and rosiglitazone andpioglitazone each upregulated TGFβR-1 expression without reachingsignificance, (FIG. 13).

7/Collagen Type I, Alpha I (Col1a) Expression

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyinhibited Col1a expression compared to vehicle (p<0.01), whereascaptopril had no effect on Col1a expression, and rosiglitazone andpioglitazone each significantly upregulated Col1a expression (p<0.001)(FIG. 14).

8/Collagen Type III, Alpha I (Col3a) Expression

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyinhibited Col3a expression compared to vehicle (p<0.001), whereascaptopril had no effect on Col3a expression, rosiglitazone significantlyupregulated Col3a expression (p<0.05), and pioglitazone upregulatedCol3a expression without reaching significance (FIG. 15).

9/Pathological Glomeruli

In anti-GBM-exposed mice, compound A (100 mg/kg/day) significantlyreduced the number of pathological glomeruli (p<0.001) (FIG. 16).

Example 3: Effect of Compound A on the Development of Bleomycin-InducedPulmonary Fibrosis in Mice, and Comparison with a Known PPARγ Agonist

Bleomycin-induced pulmonary fibrosis in mice is an in vivo modelcommonly used to evaluate the anti-fibrotic potential of new chemicalentities (Corbel et al, 2001; Manoury et al, 2006). The effect ofcompound A and rosiglitazone, a known PPARγ agonist, has accordinglybeen assessed in such a model. The C57BL/6J mouse has been chosen toevaluate the effects of the test compounds since it is prone to developan early inflammatory response followed by fibrotic remodelling in lungafter administration of bleomycin.

Mice were daily orally treated for 15 days with compound A at twodifferent doses (30 and 100 mg/kg/day) and with rosiglitazone at 5mg/kg/day. At the end of the treatment, animals were sacrificed andplasma samples and livers were harvested. Expression of genes that areknown to be implicated in the pulmonary inflammation process wasquantified and plasmatic levels of lung parameters were measured.

Bleomycin-exposed mice orally treated with vehicle for 15 days displayeda pulmonary fibrosis as shown by the statistically significant increaseof lung, osteopontin, lung MCP-1 and lung TIMP-1 levels. Treatment withcompound A at 30 and 100 mg/kg/day for 15 days significantly reducedlevels of lung TIMP-1 compared to vehicle; levels of lung MCP-1 andosteopontin were also reduced compared to vehicle without reachingstatistical significance. In contrast, when bleomycin-exposed mice wereorally daily treated with rosiglitazone at 5 mg/kg/day for 15 days, lungosteopontin, lung MCP-1 and lung TIMP-1 levels increased compared tovehicle without reaching statistical significance.

MATERIALS AND METHODS

The experiments were earned out using 77 male C57B/6J mice (JANVIERLABS, C.S. 4105, Saint-Berthevin F-53941, France), weighing 20-25 g atthe beginning of the experiment. The animals were housed in groups of3-10 in polypropylene cages (floor area=1032 cm²) under standardconditions: room temperature (22±2° C.), hygrometry (55±10%), light/darkcycle (12 h/12 h), air replacement (15-20 volumes/hour), water and food(SDS, RMI) ad libitum. Mice were allowed to accommodate themselves forat least 5 days prior to the experimentation. Mice were numbered bymarking their tail using indelible markers.

Compound A (3 mg/mL and 10 mg/mL) and rosiglitazone (0.5 mg/mL) wereformulated in 1% Methylcellulose (METOLOSE SM400, 400 Cps)+0.1%Poloxamer 188 as ready-to-use suspensions and stored at 5±3° C. duringthe study. 1% Methylcellulose (METOLOSE SM400, 400 Cps)+0.1% Poloxamer188 was used as vehicle and stored at 5±30° C. during the study.Bleomycin (Laboratoire Bellon) was dissolved in 0.9% NaCl (CDMLavoisier, France) just before use.

Dosing

Mice were allocated to the following groups:

-   -   1. 0.9% NaCl+vehicle (once a day for 15 days po), n=11,    -   2. 0.9% NaCl+compound A (100 mg/kg/day once a day for 15 days        po), n=11,    -   3. 0.9% NaCl+rosiglitazone (5 mg/kg/day once a day lot 15 days        po), n=11,    -   4. bleomycin (0.3 mg+vehicle (once a day for 15 days po), n=10,    -   5. bleomycin (0.3 mg)+compound A (30 mg/kg/day once a day for 15        days po), n=9,    -   6. bleomycin (0.3 mg)+compound A (100 mg/kg/day once a day for        15 days po), n=7,    -   7. bleomycin (0.3 mg)+rosiglitazone (5 mg/kg/day once a day for        15 days po), n=6.

On day 1, mice were anaesthetised with etomidate (15-20 mg/kg, ip) andthen intranasally administered with bleomycin sulphate (0.3 mg (300 IU)in 0.9% NaCl (50 μL/mouse (25 μL/nostril)) or with 0.9% NaCl (50μL/mouse (25 μL/nostril)). Mice were orally treated once a day for 15days (day 0 to day 14) with vehicle, compound A or rosiglitazone.Administration of vehicle, compound A or rosiglitazone on day 1 wasperformed 6 hours before 0.9% NaCl or bleomycin administration. Thevolume of administration of the test compounds was 10 mL/kg body weightfor oral administrations.

Terminal Blood sampling

On day 14, 2 hours after dosing, animals were anaesthetised withpentobarbital (60 mg/kg, ip) and blood was collected using cardiacpuncture. The exact time of dosing and time of sample collection werenoted for each animal. Blood sampling (0.9 mL of total blood) was placedin pre-chilled 2-mL lithium-heparin collection tubes. The blood sampleswere gently mixed, placed on crushed ice and centrifuged within 30 minof sampling at approximately 1500×g for 10 min at approximately +4° C.For each blood sampling, the resultant plasma was separated into 2aliquots (at least 100 μL each) and transferred using disposable plasticmaterial into polypropylene tubes. The samples were immediatelytransferred in the upright position to a freezer where they were kept at−20° C.

Lung Removal

After terminal blood sampling, lung tissue was removed:

-   -   A first tissue sample (middle lobe) was harvested and fixed with        paraformaldehyde, and kept at at 5±3° C.    -   A second tissue sample (right lobe) was frozen in liquid        nitrogen and kept at −20° C.

Measured Parameters

Collagen

For the quantification of collagen, sections were stained withPicro-Sirius red and counterstained with Mayer's haematoxylin. Allslides were digitized and 5 non overlapping fields of 3 differentsections were randomly analysed by means of the image J software(version 1.42, N.I.H., USA). For all slides, analysis was performed by asingle experimenter strictly in the same conditions.

Gene Expression

mRNA extraction was performed on small frozen lung samples (50-100 mg).Briefly, samples were cryogenically ground with mortar and pestle.Samples were subsequently homogenized using marble (2×5 mn) and 1 ml ofQiazol lysis reagent (Qiagen Ref 79306) in a Retsch MM300 apparatus. RNAextraction on lung homogenates was finalized with Qiagen Rneasy lipidKit (Ref 74804) according to the manufacturer's instructions. RNAquantity was determined with Nanodrop (ND2000 Thermo Scientific) and RNAquality was verified with Bioanalyzer (2100 Agilent Technology).

Random-primed cDNA synthesis was carried out on 100 ng total RNA usingthe Iscript kit (BIORAD ref 170-8891) according to the manufacturer'sinstructions. Real-time PCR was carried out with 7.5 ng RNA equivalentson an ABI Prism 7900 Sequence Detection System (APPLIED BIOSYSTEMS)using Iq ITaq SYBR Green Universal Rox (Ref 1725124 Biorad) and usingdedicated QPCR primers. For some mRNA transcripts, quantification wasperformed using TaqMan probes labeled with the fluorochrome FAM andusing Universal PCR MasterMix No AmpErase UNG (APPLIED BIOSYSTEMS ref4324020). The primers and probe used for the assays are listed in thefollowing tables:

Target Name Full target name Primer names Sequences (5′-3′) Rplp0ribosomal protein, Fw2Rplp0 PE ctgatgggcaagaacaccat (SEQ ID NO: 1)large, P0 Rev2Rplp0 PE gtgaggtcctccttggtgaa (SEQ ID NO: 2) Tgfbr1transforming growth MTgfbr1FW ggtcttgcccatcttcacat (SEQ ID NO: 13)factor, beta receptor 1 MTgfbr1REVcaacaggttccatttttcttca (SEQ ID NO: 14) Col1a1 collagen, type 1,MCol1a1FW aaaggtgctgatggttctcc (SEQ ID NO: 5) alpha 1 MCol1a1REVgggaccgggaggaccactgg (SEQ ID NO: 6) Col3a1 collagen, type III, MCol3a1FWgggatcaaatgaaggcgaat (SEQ ID NO: 15) alpha 1 MCol3a1REVtgggtagtctcattgccttgc (SEQ ID NO: 16) Spp1 secreted pholphoproteinMSpp1FW ctccaatcgtccctacagtc (SEQ ID NO: 17) (Osteopontin) 1 MSpp1REVggtcctcatctgtggcatca (SEQ ID NO: 18) Ccl2 chemokine (C—C motif) MCcl2FWaggtccctgtcatgcttctg (SEQ ID NO: 11) (MCP-1) ligand 2 MCcl2REVgcctactcattgggatcatc (SEQ ID NO: 12) Fn1 fibronectin 1 MFn1FWgttgtctgacgctggctttaag (SEQ ID NO: 19) MFn1REVcccacttctctccgatcttgta (SEQ ID NO: 20)

Target Name Full target name Assay ID_probe Taqman Timp-1 TIMPmetallopeptidase Mm01341361_m1 inhibitor 1

Real Time PGR was performed on ABI PRISM 7900 apparatus Raw data fromABI7900 were exported in text format. Analysis was performed on Excel,the relative quantity of transcript were calculated using the “DeltaDelta CT method” (Livak et al. Methods 2001), using Rp1p0 ashousekeeping gene for normalization and mean data from the non-treatedanimals (vehicle group) as reference control. Each RNA samples werereverse transcribed and quantified in triplicate.

Lung Protein Biomarkers

Protocol of Lung Proteins Extraction:

Extraction with T-PER®: Tissue Protein Extraction Reagent, prod#78510(lot: NG174004), Thermo Scientific with: Halt Protease InhibitorSingle-Use Cocktail. EDTA-free (100×)—Prod #78425—Lot #NL178051 ThermoFischer.

Samples of lung and supernatant were kept on ice during all experiment.For extraction, the manufacturer recommends 10 mg of tissue for 100 μlT-Per+1 μl “Halt-protease” (100×).

50 ml of T-Per buffer were prepared, to which 500 μl of “Halt-protease”(100×) were added, and the mixture was kept on ice. Samples of 50 to 70mg of lung just thawed were weighed and cut into small pieces, and 1 mlof cold PBS was added to wash the tissue. The mixture was centrifuged at500 g during 5 min at 4° C., and the supernatant was discarded. 100μl/10 mg of T-Per+antiproteases (100×) were added, crushing the lungwith a Potter, with 5 or 6 twists, up and down. The mixture wascentrifuged at 10000 g during 5 min at 4° C. The supernatant wasremoved, aliquoted and kept at −20° C. for the subsequent measurement ofbiomarkers. Samples of 10 μl were used to dose proteins by the BCAtechnique, after dilution 1/10^(ěme) in H₂O mq following the proceduredescribed in MOS: BAP-03-062-01 (Kit BCA—Pierce BCA protein assaykit—Pierce Thermo scientific, Ref: 23225).

All proteins were quantified with ELISA Kit, according to theinstructions of the manufacturers:

-   -   osteopontin: Quantikine Mouse Osteopontin immunoassay, R&D        SYSTEMS®, Ref: MOST0    -   MCP-1: Quantikine Mouse CCL2/JE/MCP-1 ELISA Kit, R&D SYSTEMS®,        ref: MJE00    -   TIMP-1: Mouse TIMP-1, R&D SYSTEMS®, Ref: TM100.

Data Processing and Statistical Analysis

All parameters were analysed using Graphpad software (version 5.1). Theparameters were analysed as followed:

-   -   Using one-way ANOVA (treatment) to compare groups (1, 2 and 3).        When ANOVA was found significant, a Dunnett's test was used to        compare group 1 to group 2 and to group 3.    -   Using one-way ANOVA (treatment) to compare groups (1, 4, 5, 6        and 7). When ANOVA was found significant, a Dunnett's test was        used to compare group 1 to group 4, to group 5, to group 6 and        to group 7).

In FIGS. 17 to 27, * denotes a p-value<0.05; ** denotes a p-value<0.01;*** denotes a p-value<0.001.

RESULTS

1/Collagen Deposition

In bleomycin-exposed mice compound A (100 mg/kg/day) significantlydecreased collagen deposition levels compared to vehicle (p<0.05).Rosiglitazone also significantly decreased collagen levels (p<0.05)(FIG. 17).

2/Lung TIMP-1

In bleomycin-exposed mice, compound A (30 and 100 mg/kg/day)significantly reduced TIMP-1 levels compared to vehicle (p<0.05) whereasrosiglitazone increased TIMP-1 levels without reaching significance(FIG. 18).

3/Lung MCP-1

In bleomycin-exposed mice, compound A (30 and 100 mg/kg/day) reducedMCP-1 levels compared to vehicle without reaching significance whereasrosiglitazone increased MCP-1 levels without reaching significance (FIG.19).

4/Lung Osteopontin

In bleomycin-exposed mice, compound A (30 and 100 mg/kg/day) reducedosteopontin levels compared to vehicle without reaching significancewhereas rosiglitazone increased osteopontin levels without reachingsignificance (FIG. 20).

5/TGFβR-1 Expression

In bleomycin-exposed mice, compound A (100 mg kg day) significantlyinhibited TGFβR-1 expression compared to vehicle (p<0.05). Rosiglitazonealso significantly inhibited TGFβ-1 expression (p<0.05) (FIG. 21).

6/Collagen Type I, Alpha I (Col1a) Expression

In bleomycin-exposed mice, compound A (100 mg/kg/day) significantlyinhibited Col1a expression compared to vehicle (p<0.001) whereasrosiglitazone upregulated Col1a expression without reaching significance(FIG. 22).

7/Collagen Type III, Alpha I (Col3a) Expression

In bleomycin-exposed mice, compound A (100 mg/kg/day) significantlyinhibited Col3a expression compared to vehicle (p<0.001) whereasrosiglitazone inhibited Col3a expression without reaching significance(FIG. 23).

8/TIMP-1 Expression

In bleomycin-exposed mice, compound A (30 and 100 mg/kg/day) inhibitedTIMP-1 expression compared to vehicle without reaching significancewhereas rosiglitazone significantly upregulated TIMP-1 expression(p<0.05) (FIG. 24).

9/MCP-1 Expression

In bleomycin-exposed mice, compound A (30 and 100 mg/kg/day) inhibitedMCP-1 expression compared to vehicle without reaching significancewhereas rosiglitazone upregulated TIMP-1 expression without teachingsignificance (FIG. 25).

10/Osteopontin Expression

In bleomycin-exposed mice, compound A (30 and 100 mg kg/day)significantly inhibited osteopontin expression compared to vehicle(p<0.05) whereas rosiglitazone had no effect on osteopontin (FIG. 26).

11/Fibronectin Expression

In bleomycin-exposed mice, compound A (100 mg/kg/day) significantlyinhibited fibronectin expression compared to vehicle (p<0.05) whereasrosiglitazone upregulated fibronectin expression without reachingsignificance (FIG. 27).

The above results show that oral administration of compound A (30 or 100mg/kg/day) in male C57BL/6J mice reduced the increase of inflammatoryfibrotic biomarkers levels induced by bleomycin instillation in lungwhereas rosiglitazone had no effect, or even a detrimental effect onthese biomarkers. Taken together, these data show that compound Areduced bleomycin-induced pulmonary fibrosis in mice.

Example 4: Effect of Compound A on the Development of Bleomycin-InducedSkin Fibrosis in Mice, and Comparison with a Known PPARγ Agonist

Compound A was tested in a murine model of bleomycin-induced skinfibrosis. Mice were daily orally treated for 21 days with compound A attwo different doses (30 and 100 mg/kg/day) and with the PPARγ referencecompound rosiglitazone at 5 mg/kg/day. At the end of the treatment,animals were sacrificed and skin samples were taken. Expression of genesthat are known to be implicated in the systemic fibrosis pathway wasquantified and dermal thickness and collagen content was determined.

Bleomycin-exposed mice orally treated with vehicle for 21 days displayedskin fibrosis as shown by the statistically significant increase ofdermal thickness and collagen content. Treatment with compound A at 50and 100 mg/kg/day for 21 days significantly reduced dermal thickness andcollagen content compared to vehicle. A similar effect was observed inbleomycin-exposed mice orally daily treated with rosiglitazone at 5mg/kg/day for 21 days, although the effect was less marked regardingdermal thickness.

MATERIALS AND METHODS

The experiments were carried out on 6-week-old male C56BL/6 mice(Janvier, Le Genest-Saint-Isle, France). The animals were housed ingroups of 3-10 in polypropylene cages (floor area=1032 cm²) understandard conditions: room temperature (22±2° C.), hygrometry (55±10%),light/dark cycle (12 h/12 h), air replacement (15-20 volumes/hour),water and food (SDS, RM1) ad libitum. Mice were allowed to accommodatethemselves for at least 5 days prior to the experimentation. Mice werenumbered by marking their tail using indelible markers.

Compound A (3 mg/mL and 10 mg/mL) and rosiglitazone (0.5 mg/mL) wereformulated in 1% Methylcellulose (METOLOSE SM400, 400 Cps)+0.1%Poloxamer 188 as ready-to-use suspensions and stored at 5±3° C. duringthe study. 1% Methylcellulose (METOLOSE SM400, 400 Cps)+0.1% Poloxamer188 was used as vehicle and stored at 5±3° C. during the study.

Dosing

Mice were allocated to the following groups:

-   -   1. 0.9% NaCl+vehicle (once a day for 21 days po), n=7,    -   2. 0.9% NaCl+compound A (100 mg/kg/day once a day for 21 days        po), n=8,    -   3. 0.9% NaCl+compound A (30 mg/kg/day once a day for 21 days        po), n=8,    -   4. bleomycin (0.3 mg)+rosiglitazone (5 mg/kg/day once a day for        21 days po), n=8,    -   5. bleomycin (0.3 mg)+vehicle (once a day for 21 days po), n=6,    -   6. bleomycin (0.3 mg)+compound A (100 mg/kg/day once a day for        21 days po), n=7,    -   7. bleomycin (0.3 mg)+compound A (30 mg/kg/day once a day for 21        days po),

Skin fibrosis was induced by daily injections of bleomycin (100 μL ofbleomycin (Laboratoire Bellon, France) dissolved in 0.9% NaCl (CDMLavoisier, France) at a concentration of 0.5 mg/ml, administered 6days/week into defined areas of 1 cm² on the upper back). 0.9% NaCl wasused as control (100 μL subcutaneous injections).

Mice were orally treated once a day for 21 days with vehicle, compound Aor rosiglitazone. Administration of vehicle, compound A or rosiglitazoneon day 1 was performed 4 hours before 0.9% NaCl or bleomycinadministration.

Skin Sampling

On day 21, mice were sacrificed by cervical dislocation, and skinsamples were taken and processed for analysis.

Measured Parameters:

Dermal and Adipose Layer Thickness

Lesional skin areas were excised, fixed in 4% formalin and embedded inparaffin. Five μm thick sections were stained with haematoxylin andeosin. The dermal thickness was analyzed at 100-fold magnification bymeasuring the distance between the epidermal-dermal junction and thedermal-subcutaneous fat junction at four sites from lesional skin ofeach mouse. Two independent examiners performed the evaluation before aconsensus in case of more of 10% of variability of the measures.

Collagen Content of Skin

Hydroxyproline Assay

The collagen content in lesional skin samples was explored byhydroxyproline assay. After digestion of punch biopsies (Ø 3 mm) in 6MHCl for three hours at 120° C., the pH of the samples was adjusted to 7with 6M NaOH. Afterwards, samples were mixed with 0.06 M chloramine Tand incubated for 20 min at room temperature. Next, 3.15M perchloricacid and 20% p-dimethylaminobenzaldehyde were added and samples wereincubated for an additional 20 min at 60° C. The absorbance wasdetermined at 557 nm with a Spectra MAX 190 microplate spectrophotometer(Molecular Devices, Sunnyvale, Calif., USA).

Sircol Assay

Total soluble collagen in cell culture supernatants was quantified usinga Sircol collagen assay (Biocolor). Briefly, cell culture supernatantwas mixed with sirius red dye for 30 minutes at room temperature. Aftercentrifugation, the pellet was dissolved in alkali reagent. Measurementwas performed using a SpectraMax 190 microplate spectrophotometer(Molecular Devices) at a wavelength of 540 nm.

Data Processing and Statistical Analysis

Data were expressed as mean±standard error of the mean. The Studentt-test was used for statistical analyses. A p value of less than 0.05was considered as a statistically significant result.

In FIGS. 28 to 30 * denotes a p-value≤0.05; ** denotes a p-value<0.01;*** denotes a p-value<0.001.

RESULTS

Injection of bleomycin in mice resulted in an increase in dermalthickness compared to mice receiving NaCl (see FIG. 28: increase of 57%in the group bleomycin+vehicle compared to the group NaCl+vehicle).Rosiglitazone and compound A (30 mg/kg) significantly reduced dermalthickness in bleomycin-exposed mice compared to vehicle (p≤0.05).Compound A (100 mg/kg) more significantly reduced dermal thickness inbleomycin-exposed mice compared to vehicle (p<0.001).

Consistent with the reduced dermal thickening, the hydroxyprolinecontent in lesioned skin of mice treated with compound A (30 and 100mg/kg) and rosiglitazone was significantly lower than in lesioned skinof mice treated with vehicle, compound A (30 mg/kg) being more efficientthan rosiglitazone (FIG. 29).

Compound A (30 and 100 mg/kg) and rosiglitazone also significantlyreduced the collagen content compared to vehicle (FIG. 30).

Altogether these results show that compound A is efficient to preventskin fibrosis. Compound A (100 mg/kg) even exerts better effects ondermal thickness compared to rosiglitazone.

Example 5: Activation of Human and Murine PPAR Receptors by Compound A

The ability of compound A to activate all three subtypes of PPARreceptors was determined by transient transactivation assays. Thesecell-based assays were carried out using Cos-7 cells transfected with achimeric human or mouse PPARα-Gal4 receptor expression plasmid (orPPARδ-Gal4, or PPARγ-Gal4) and a 5Gal4 pGL3 TK Luc reporter plasmid.Transfections were performed by a chemical agent (Jet PEI). Transectedcells were distributed in 384-wells plates and were allowed to recoverfor 24 h. The culture medium was then removed and fresh mediumcontaining the compound to be tested (5 μM) was added (finalconcentration ranging from 10⁻⁴ M to 3 10⁻¹⁰ M). After an overnightincubation, luciferase expression was measured by adding SteadyGloaccording to the manufacturer's instructions (Promega). Fenofibric acidat 10⁻⁵M, GW501516 at 10⁻⁸M. and Rosiglitazone at 10⁻⁶ M were used asreferences. Results were expressed as percentage activity compared toreferences (fenofibric acid for PPARα, rosiglitazone for PPARγ, andGW501516 for PPARδ) taken as 100%. For human receptors, the results arethe mean of 6 experiments, each in quadruplicate. For murine receptors,the results are the mean of 5 (PPARδ) or 6 (PPARα and γ) experiments,each in quadruplicate. Dose-effect curves and EC50 were calculated usingthe software Assay Explorer (MDL). The results are presented in thetable below and in FIGS. 31 and 32.

PPARα PPARγ PPARδ human mouse human mouse human mouse EC50 (μM) 0.920.29 0.18 0.17 0.53 2* *estimated (plateau not reached)

These results show that compound A activates all three subtypes of PPARreceptors with an EC50 of less than 1 μM for each subtype. It canfurther be seen that compound A has a balanced activity between thethree subtypes of PPAR receptors.

Overall, the results of examples 1-5 suggest that a good PPARδ agonistactivity is required alongside with PPARα and PPARγ agonist activitiesin order in exert a pan-antifibrotic effect.

Example 6: Effect of Compound A on Proliferation of Lung and DermalFibroblasts, and Comparison with Known PPARγ and PPARα Agonists

The ability of compound A to inhibit PDGF-induced lung and dermalfibroblasts proliferation was determined by performing EdU(5-ethynyl-2′-deoxyuridine) incorporation assays. These assays werecarried out using primary human lung or skin fibroblasts (Promocell).The cells were plated in 96-well plates in full growth medium for 24 h,followed by 24 h starvation in serum-free medium. The medium was thenreplaced by fresh medium containing PDGF and compound A to be tested (atconcentrations ranging from 10⁻⁴ M to 4.5 10⁻⁸ M) for another 24 h EdUwas added to the cells for the last 16 h of the compound treatment. Theculture medium was then removed, the cells were fixed with formaldehydeand the EdU incorporated in the DNA of diving cells was quantified usingfluorescent Click-it assay according to the manufacturer's instructions(Invitrogen). Fenofibric acid at concentrations ranging from 3 10⁻⁴ M to1.4 10⁻⁷ M (PPARα) and Rosiglitazone at concentrations ranging from 310⁻⁵ M to 1.4 10⁻⁸ M (PPARγ) were used as references. Results wereexpressed as % of EdU-positive cells out of the total cell number. Theresults present the mean of biological triplicates. Dose-effect curvesand IC₅₀ values were calculated using the GraphPad Prism software. Theresults are presented in the table below and in FIGS. 33 and 34.

compound A fenofibric acid rosiglitazoneγ Lung Dermal Lung Dermal LungDermal IC₅₀ (μM) 10.90 11.50 NC NC NC NC NC: not converged

Example 7: Effect of Compound A on Fibroblasts to MyofibroblastsTransition of Lung and Dermal Fibroblasts, and Comparison with KnownPPARγ and PPARα Agonists

The ability of compound A to inhibit TGFβ-induced lung and dermalfibroblasts to myofibroblasts transition (FMT) was determined byperforming immunocytochemistry assays for the myofibroblast marker,α-smooth muscle actin (α-SMA). These assays were carried out usingprimary human lung or skin fibroblasts (Promocell). The cells wereplated in 96-well plates in full growth medium for 24 h, followed by 24h starvation in serum-free medium. The medium was then replaced by freshmedium containing TGFβ and compound A to be tested (at concentrationsranging from 10⁻⁴ M to 4.5 10⁻⁸ M) for another 48 h. The culture mediumwas then removed, the cells were fixed with formaldehyde and stainedwith a primary mouse α-SMA antibody (Sigma) and secondaryfluorescence-labelled goat-anti-mouse antibody (Invitrogen). α-SMAexpression was quantified using Meta Xpress software. Fenofibric acid atconcentrations ranging from 3 10⁻⁴ M to 1.4 10⁻⁷ M (PPARα) andRosiglitazone at concentrations ranging from 3 10⁻⁵ M to 1.4 10⁻⁸ M(PPARγ) were used as references. Results were expressed as % ofα-SMA-positive cells out of the total cell number. The data wasnormalised to the TGFβ treatment alone, which was taken as 100%. Theresults present the mean of biological triplicates.

Dose-effect curves and IC₅₀ values were calculated using the GraphPadPrism software. The results are presented in the table below and inFIGS. 35 and 36.

compound A fenofibric acid rosiglitazone Lung Dermal Lung Dermal LungDermal IC₅₀ (μM) 10.79 ~11.18 NC NC NC NC NC: not converged

Altogether, these in vitro functional data demonstrate that compound Aefficiently inhibits PDGF-induced proliferation and TGFβ-inducedmyofibroblasts transition in primary human lung and dermal fibroblasts,thus providing a link with the anti-fibrotic effects observed in vivo.In addition, these results suggest that pan-PPAR agonism might besuperior to a single PPAR activation in its anti-fibrotic effects in thetarget cells on the two key fibrogenic pathways.

The invention claimed is:
 1. A method of treating a fibrotic conditionwhich comprises administering a therapeutically effective amount of apan-PPAR agonist to a human subject in need thereof, wherein thepan-PPAR agonist is5-chloro-1-[(6-benzothiazolyl)sulfonyl]-1H-indole-2-butanoic acid. 2.The method of claim 1, wherein the pan-PPAR agonist is administeredorally.
 3. The method of claim 2, wherein the pan-PPAR agonist isadministered in the form of a pharmaceutical composition comprising oneor more pharmaceutically acceptable excipients.
 4. The method of claim3, wherein the pharmaceutical composition is a tablet, a capsule or alozenge.
 5. The method of claim 3, wherein the pharmaceuticalcomposition comprises from about 10 mg to about 1000 mg of said pan-PPARagonist.
 6. The method of claim 1, wherein the pan-PPAR agonist isadministered parenterally.
 7. The method of claim 6, wherein thepan-PPAR agonist is administered in the form of an aqueous solutioncomprising one or more pharmaceutically acceptable excipients.
 8. Themethod of claim 1, wherein the pan-PPAR agonist is administered byinhalation.
 9. The method of claim 1, wherein the pan-PPAR agonist isadministered intranasally.
 10. The method of claim 1, wherein thepan-PPAR agonist is administered topically.